Abstract

Background There is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models.

Methods Myocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia–reperfusion in Sprague–Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks after injection. Whole transcriptome microarrays were performed on RNA isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histologic quantification confirmed expression of key genes and their activation in altered pathways.

Conclusions These results indicate that the myocardial matrix alters several key pathways after MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

Progression from acute myocardial infarction (MI) to chronic heart failure (HF) begins with an initial ischemic injury, resulting in progressive myocyte loss through both necrotic and apoptotic mechanisms, and migration of inflammatory cells into the injured myocardium. An increase in matrix metalloproteinases from the inflammatory infiltrate further exacerbates the decline in heart function by digesting the extracellular matrix (ECM), followed by subsequent deposition of fibrillar cross-linked collagen. The heart has recently been recognized as an organ capable of some degree of self-regeneration (1). However, this is insufficient to compensate for the billions of cardiomyocytes lost after MI (2). Additionally, function of surviving cardiomyocytes is also altered after infarction. The heart has a high energy demand and recent studies have shown that dysregulation in cardiac metabolism after MI contributes notably to cardiac dysfunction in HF (3).

Interest in developing alternative treatments for MI has been expanding. Such therapies include various cells, biological molecules, acellular biomaterials, or combinations thereof. Meta-analyses of initial cell therapy trials suggest only a modest effect on cardiac function (4), and given low cell survival rates and their largely paracrine mechanism of action, there has been increasing interest in the use of injectable acellular scaffolds (5). If designed appropriately, these biomaterials can be delivered through minimally invasive approaches and stimulate cardiac repair, while avoiding many of the complications associated with a living product (6). Our group previously developed an injectable myocardial matrix hydrogel, derived from decellularized porcine ventricular ECM (7), which can be delivered with a transendocardial catheter. This hydrogel was shown to reduce negative left ventricular (LV) remodeling and the decline in cardiac function in both rat (8) and pig (9) models when delivered 2 weeks after MI. Herein, we examined whether the material could improve global cardiac function and hemodynamics when delivered 1 week after MI in a rat model, and utilized a transcriptomics-directed approach to identify the underlying mechanisms by which the matrix improves post-MI repair.

Methods

All procedures in this study were approved by the Committee on Animal Research at the University of California, San Diego, and the Association for the Assessment and Accreditation of Laboratory Animal Care. Myocardial matrix or saline was injected into the area of ischemia 1 week after 25 min of ischemia–reperfusion in female Sprague-Dawley rats. Rat Gene 2.0 ST arrays (Affymetrix, Inc., Santa Clara, California) were used for whole transcriptome analysis of infarct and border zone at 3 days and 1 week after injection (see Online Figure 1 for a representative H&E slide from infarcted hearts that was included for analysis), followed by validation of the expression of key genes by quantitative real-time polymerase chain reaction (qPCR, primer sequences in Online Table 1). Cardiac magnetic resonance imaging and hemodynamics recordings were performed at 5 weeks after injection (6 weeks after MI). Histology and immunohistochemistry (IHC) were used to quantify phenotypic changes. For further details, refer to the Online Appendix.

Transcriptomics

Differences in transcriptomes between saline- and matrix-treated infarcts were globally examined using both principal component analysis and hierarchical clustering. Saline- and myocardial matrix-injected samples did not cluster separately at 3 days after injection. However, by 1 week after injection, both principal component analysis (Figure 2A) and hierarchical clustering (Figures 2B and 2C) showed separation of the transcriptomes (p = 0.06), indicating a shift in global gene expression. A false discovery rate of q < 0.05 was used to determine the differentially expressed genes for biological interpretation (Online Table 3). A Panther overrepresentation test showed that differences in transcripts from ECM (Gene Ontology [GO]: 0031012) and those responsible for muscle contraction (GO:006936) for the 1-week time point were nonsignificant (p = 0.37 and p = 0.88, respectively), indicating that differences in gene expression were not due to sampling variability. Using Ingenuity Pathway Analysis (IPA) (Qiagen, Redwood City, California), we identified the main effects of the myocardial matrix at 3 days after injection as downregulation of apoptosis, upregulation of blood vessel development, and increase in cell movement (Online Table 4). By 1 week, several pathways were significantly activated, including downregulation of cell death and hypertrophy, and upregulation of many metabolic processes and gene translation/transcription (Online Table 5). Further details, as well as the complete list of differentially expressed genes, can be found in Online Tables 6 through 13. Based on the differences elucidated by this transcriptome analysis, we performed qPCR on key genes in the identified pathways (Online Figure 2) and further assessed differences at the tissue level using IHC as described later.

Inflammatory Response

Pathways involved in the immune response including migration and infiltration of various cell types were predicted at both the 3-day and 1-week time points (Online Tables 4 and 5, Figure 3A, Online Figure 3). Similarly, a substantial number of genes at both time points —26.6% at day 3 and 9.8% at 1 week—were characterized as part of immune response process (GO:0002376). Increased expression of CD68 (p = 0.045) and matrix metalloproteinase 12 (p = 0.043) (Online Figure 2A) were confirmed by qPCR, indicative of increased macrophage infiltration as a result of matrix injection, yet IHC analysis using a CD68 antibody (Figure 3B) did not show a difference in macrophage infiltration between saline- and matrix-injected infarcts at 3 days (Figure 3C). There was, however, a trend toward an increase in infiltrating tryptase+ mast cells (Figure 3D) at 3 days (p = 0.052), which reached significance by 1 week (p = 0.032) (Figure 3E).

Blood Vessel Formation

Activation of blood vessel development was predicted by IPA at 3 days after injection (Online Table 4, Figure 4A). Although increased vessel development was not directly predicted at 1 week after injection, many growth factors associated with angiogenesis and neovascularization were identified (Figure 4A). These included a decrease in angiopoietin-2 (p = 0.012) and increases in acidic fibroblast growth factor (p = 0.053) and vascular endothelial growth factors A and B (p = 0.034 and p = 0.009, respectively), confirmed by qPCR (Online Figure 2B). Infarct vascularization was then examined by IHC (Figure 4B). Although not different at 3 days after injection, capillary density was significantly greater in the matrix-injected group at 1 week (p = 0.038) (Figure 4C). Arteriole density showed a similar result, with no difference at 3 days, but there was a trend toward an increase at 1 week in the matrix group (p = 0.056) (Figure 4D). When subdivided into large (Online Figure 4A), medium (Online Figure 4B), and small (Online Figure 4C) arterioles, there was a trend at 3 days for increased small arterioles within matrix-injected infarcts (p = 0.082), which shifted at 1 week to a significant increase in medium (p = 0.021) and a trend for an increase in large diameter vessels (p = 0.065).

Cardiomyocyte Apoptosis

Decreased apoptosis was predicted consistently by IPA at both 3 days and 1 week after injection (Online Tables 4 and 5, Figure 5A, Online Figures 5 and 6). Increased expression of antioxidative enzymes heme oxygenase 1 (p = 0.015) at 3 days and catalase (p = 0.002) at 1 week, as well as the antiapoptosis regulator Bcl-2 (p = 0.006) at 1 week, were confirmed using qPCR (Online Figure 2C). To determine the effect that myocardial matrix injection may have on apoptosis of cardiomyocytes specifically, anti–cleaved-caspase 3 staining with colabeling of cardiomyocytes using α-actinin was performed (Figure 5B). Quantification of the number of caspase-3–expressing cardiomyocytes within the infarct wall showed a trend toward decreased apoptotic cardiomyocytes within the infarct wall (p = 0.085) at 3 days after injection (Figure 5C).

Discussion

Previous small and large animal studies have demonstrated that injection of acellular myocardial matrix improved cardiac function and reduced negative LV remodeling when delivered 2 weeks after MI. Importantly, biocompatibility, hemocompatibility, and lack of arrhythmias were demonstrated (8,9). In the current study, we analyzed infarct gene expression using whole-transcriptome microarrays to gain a comprehensive understanding of the tissue-level mechanism of action of the myocardial matrix hydrogel. Gene expression was analyzed at both 3 days and 1 week after injection based on previous studies indicating that cellular infiltration into the injected hydrogel was most pronounced during the first week (9). By 1 week after injection, the transcriptomes of saline and matrix-injected infarcts clustered separately. Similar analyses have also been applied to other experimental therapies for MI including cell transplantation (10–12) and injection of cell-derived products (13); however, to our knowledge, no other biologic-based therapy has been reported to induce a distinct transcription signature at a global level. In this study, the key modulated pathways included inflammation, reduction of apoptosis and cardiac hypertrophy, metabolism, and blood vessel and cardiac development (Central Illustration).

Injection of myocardial matrix 1 week after myocardial infarction (MI) into the infarcted area induced various tissue level changes that reduced negative left ventricular remodeling and improved hemodynamics. Altering these key pathways created a pro-regenerative environment, potentially preventing or slowing development of heart failure.

Gene expression differences within the infarct suggested an increase in macrophage migration in response to the matrix injection with increased transcription of CD68, a macrophage marker, and matrix metalloproteinase 12, a macrophage-specific protease (14). However, differences in transcription patterns could also be attributed to changes in immune cell behavior, because an increase in macrophages was not demonstrated. Collective analysis of all differentially expressed transcripts related to inflammation was not conclusive regarding whether there was a predominance of either M1 or M2 macrophage activation, which has been attributed to proinflammatory and pro-remodeling responses, respectively (15). Both phenotypes are likely necessary for postinfarct repair as depletion of either M1 or M2 macrophages inhibited the ability of neonatal hearts to regenerate after MI (16). In the process of identifying c-Kit+ progenitors, which stain negative for tryptase, we detected a notable increase in the number of tryptase+ mast cells in the matrix-injected groups. Although mast cells are traditionally associated with an allergic response, they are also involved in neovascularization and regulation of the immune response (17). After ischemia, rapid mast cell degranulation occurs, triggering recruitment of other leukocytes and preventing cardiomyocyte apoptosis (18). Additionally, mast cell products are known to be inherently angiogenic and stimulate endothelial secretion of angiogenic chemokines (19). Although it is known that mast cell activation may be the first step in the acute inflammatory response to implanted biomaterials (20), implication on the reparative response of decellularized materials has not been reported.

By 1 week after injection, myocardial matrix had also significantly increased infarct neovasculature. Interestingly, the changes over time seemed to be due to a decrease in endothelial cells and arterioles in the saline-injected control subjects. Immediately after MI, hypoxia-inducible factor expression triggers transcriptional activation of many angiogenic factors (21); however, a decrease in vascular density with time has been previously reported (22), possibly due to vessel regression. Notably, 1 factor known to induce this process is angiopoietin 2, which was more highly expressed in the saline group compared with the matrix. Preservation of the infarct vasculature may be a result of the pro-angiogenic milieu induced by the myocardial matrix, whether indirectly, through its effects on other cell types such as immune cells, or directly, by creating a new physical scaffold for vessel infiltration or releasing bioactive matricryptic peptides from partial proteolysis of the ECM (23).

Pathway analysis from the microarray data predicted consistent downregulation of apoptosis and cell death. Specifically, matrix-injected infarcts expressed higher levels of heme oxygenase 1 at 3 days and catalase at 1 week, both of which are stress-induced enzymes that reduce reactive oxygen species. In the histologic analysis, we demonstrated a trend toward a reduction in cardiomyocyte apoptosis in the infarct wall. After an infarct, cardiomyocyte death peaks 24 h after the injury then decreases, but continues to be increased above baseline levels for at least 12 weeks (24). Therefore, delivery of the myocardial matrix could play an important role in salvaging cardiomyocytes that are pre-apoptotic. Pathway analysis also predicted upregulation of genes involved in mitochondrial metabolism. It is well-demonstrated that myocardial ischemia reduces substrate oxidation, resulting in an increased reliance on glycolysis (25). After matrix injection, we found increased expression of PGC-1α, PPARα, PPARβ/δ, and estrogen-related receptor-γ. The PGC-1α activates the transcription of these nuclear receptors to increase fatty acid uptake, oxidative phosphorylation, and mitochondrial biogenesis (26). Expression of PGC-1α targets is known to be downregulated in both rodent models and patients with HF (27). We showed an increase in PGC-1α in cardiomyocytes in matrix-injected animals. In comparison, injections of various bone marrow cells into the infarct myocardium have been associated with decreased expression of genes related to mitochondrial function (10,12). Angiotensin-converting enzyme inhibition using captopril, a common treatment for HF, similarly did not rescue changes in fatty acid metabolism (28).

The adult mammalian heart has a limited ability to regenerate, and many recent efforts have attempted to enhance this ability after MI (2). GO analysis suggested activation of terms related to heart and cardiovascular system development, which led us to investigate whether there was an upregulation of cardiac transcription factors known to play a role in cardiac regeneration. We found increased expression of 6 cardiac transcription factors. Of these, GATA4, myocardin, Nkx2.5, Tbx5, and Tbx20 are all expressed throughout various stages of embryonic cardiogenesis (29), whereas GATA4, Nkx2.5, and Tbx5 are frequently used to identify various CPC subsets (30). Increased expression of MEF and GATA4 were reported after injection of bone marrow cells and mononuclear cell secretomes (10,13); however, to our knowledge, concomitant increased transcription of several cardiac transcription factors has not been reported previously. A lineage tracing study will, however, be necessary to determine whether the myocardial matrix can induce cardiac regeneration, because increased expressions of these factors can occur during other processes. For example, GATA4, myocardin, Nkx2.5, and Tbx20 are expressed by adult cardiomyocytes and are required for their survival and function (31–34); cardiac hypertrophy is associated with elevated GATA4, MEF2d, and Nkx2.5 (35,36); and cardiac fibroblasts can express high levels of GATA4 and Tbx20 (37). We, however, show an increase of 6 transcription factors for cardiac development, along with a decrease in cardiac hypertrophy and fibrosis. We also found a significant increase in ckit+/tryptase-CPCs in matrix-injected hearts. Previously, the myocardial matrix was shown to promote cardiac differentiation of cKit+ CPCs in vitro (38). In this study, we found some cKit+ cells coexpressing Nkx2.5, which has been used to identify cardiac lineage differentiation in vivo (39). The importance of c-kit+ cells in post-infarct regeneration, however, has been a controversial subject and 2 recent studies arrived at opposite conclusions (40,41). Additionally, cKit+ cells may contribute to other effects of the myocardial matrix injection, such as neovascularization (41).

Previous studies with myocardial matrix tested delivery at 2 weeks after MI (8,9). In the current study, we injected the material 1 week after MI, demonstrating that the matrix can also be effective at an earlier time point. Hemodynamics, which had not been studied previously, further demonstrated improvements in LV peak systolic pressure, myocardial contractility, and myocardial relaxation. As further evidence that the matrix attenuates negative LV remodeling, decreased hypertrophy was suggested by transcriptional analysis as early as 1 week after injection, and there was a trend toward a reduction in cardiomyocyte area and a significant decrease in interstitial fibrosis by 5 weeks.

Study Limitations

Limitations of this study include the use of a small animal model, relatively short time points of assessment after injection, and the more mild ischemia–reperfusion model, which is likely more representative of the acute or subacute MI population rather than severe remodeling in HF patients. Although changes in cardiac function were significant in this and a previous rat MI study, with similar injections and time points (8), greater increases in function were observed in a large animal model where the infarct was more severe, multiple injections were performed across the infarct, and cardiac function was examined out to 3 months after injection (9). Unlike cells or growth factors, which have had diminished efficacy in moving from small to large animals, biomaterials may have the capacity for greater improvements in larger animals (42), which is promising for future clinical translation. This study also tested a porcine-derived ECM hydrogel in a rat model, which has potential for xenograft-elicited inflammation. However, we observed similar improvements in a porcine MI model with the porcine-derived material (9), suggesting that the interspecies effects are not a major factor. Also, this study mimicked the xenogeneic porcine material source being used in an ongoing clinical trial (NCT02305602).

Conclusions

We demonstrated decreased negative LV remodeling and improved hemodynamics following delivery of myocardial matrix 1 week after MI. We provided both transcriptional and histologic evidence that the myocardial matrix mediated this by inducing various tissue level changes. These results provide further evidence for the promise of the myocardial matrix as a therapy to prevent development of HF after MI.

Perspectives

COMPETENCY IN MEDICAL KNOWLEDGE: In animal models, injection of a hydrogel derived from decellularized porcine ventricular myocardium attenuated the decline in cardiac function after MI.

TRANSLATIONAL OUTLOOK: Clinical trials are needed to evaluate the safety and efficacy of injectable hydrogels prepared from myocardial extracellular matrix in patients with MI.

Acknowledgments

The authors thank Dr. Nicholas Webster of the San Diego VA/VAMF Microarray and NGS Core for assistance with microarray analysis as well as Dr. Christian Metallo for helpful discussions regarding metabolism.

Appendix

Appendix

For an expanded Methods section, and supplemental tables, figures, and references, please see the online version of this article.

Footnotes

This research was funded in part by the National Institutes of Health National Heart, Lung, and Blood Institute (1R01HL113468). Ms. Wassenaar was supported by pre-doctoral fellowships from the California Institute for Regenerative Medicine and the American Heart Association, as well as the University of California, San Diego Medical Scientist Training Program T32 GM007198-40. Dr. DeMaria is a scientific advisory board member of Ventrix, Inc. Dr. Christman is a co-founder, board member, consultant, and holds equity interest in Ventrix, Inc. All other authors have reported that they have no relationships relevant to the contents of this paper disclose.

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